The pyrazole compound LQFM-021 exhibits vasorelaxant, antinociceptive and anti-inflammatory activities. Furthermore, it has low toxicity, indicating that this compound may be considered to be a good ...prototype for the development of new analgesic/anti-inflammatory drugs. Therefore, the aim of this study was to investigate the potential anti-inflammatory activity of LQFM-021 using a model of carrageenan-induced inflammation as well as the mechanism of action and role of nitric oxide in this effect. Acute treatments with LQFM-021 (30 and 60 mg/kg p.o.) reduced paw edema formation dose-dependently 2 h after carrageenan injection. In the carrageenan-induced pleurisy test, LQFM-021 (30 mg/kg p.o.) reduced the leukocyte (polymorphonuclear) count in the pleural cavity, as well as decreased protein extravasation and myeloperoxidase activity. This dose of LQFM-021 increased the NO (nitrite/nitrate) and IL-4 levels and decreased the TNF-α and IL-1β levels in the pleural cavity. Moreover, pre-treatment with L-NAME reversed the effect of LQFM-021 on NO, leukocyte migration, and the TNF-α and IL-1β levels. Additionally, we observed that LQFM-021 showed weak inhibitory activity on cyclooxygenases, but reduced the PGE2 levels in the pleural cavity. Immunoblot analyses showed that LQFM-021 promoted a decrease in COX-2 levels and increase in iNOS levels. In conclusion, we demonstrated that LQFM-021 has marked anti-inflammatory activity by reducing polymorphonuclear recruitment, which is associated with the inhibition of the production of inflammatory cytokines and eicosanoids. In addition, we found that the synthase/release of nitric oxide promoted by LQFM-021 is essential for the anti-inflammatory effect observed.
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•Anti-inflammatory activity of LQFM-021 in carrageenan-induced inflammation models.•LQFM-021 inhibition of the production of inflammatory cytokines and eicosanoids.•LQFM-021 increased the NO (nitrite/nitrate) in pleural exudate.•Role of nitric oxide in the anti-inflammatory activity of LQFM-021.
To perform the proteomic profile of Paracoccidioides lutzii after treatment with the compound camphene thiosemicarbazide (TSC-C) in order to study its mode of action.
Proteomic analysis was carried ...out after cells were incubated with TSC-C in a subinhibitory concentration. Validation of the proteomic results comprised the azocasein assay, western blot and determination of the susceptibility of a mutant to the compound.
Proteins related to metabolism, energy and protein fate were regulated after treatment. In addition, TSC-C reduces the proteolytic activity of the protein extract similarly to different types of protease inhibitors.
TSC-C showed encouraging antifungal activity, working as a protease inhibitor and downregulating important pathways impairing the ability of the fungi cells to produce important precursors.
The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs ...and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. Of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.
Considering the need to identify new compounds with antifungal action, the activity of five 3-phenacylideneoxindoles compounds was evaluated.
The compounds were synthesized, and their antifungal ...activity was elucidated through minimum inhibitory concentration tests and interaction assay with other antifungals. Potential targets of compounds were predicted
.
3-phenacylideneoxindoles compounds inhibited fungal growth with minimum inhibitory concentration and minimum fungicidal concentration ranging from 3.05 to 12.26 μM. The compounds demonstrated high selectivity index and presented a synergistic effect with itraconazole.
prediction revealed the pentafunctional AROM polypeptide, enolase, superoxide dismutase, catalase and kinases as proteins targets of the compound 4a.
The results demonstrate that 3-phenacylideneoxindoles is a potential new class of antifungal compounds for paracoccidioidomycosis treatment.
As part of our continuing chemical and biological analyses of Rubiaceae species from Cerrado, we isolated novel alkaloids 1 and 2, along with known compounds epicatechin, ursolic acid, and oleanolic ...acid, from Galianthe ramosa. Alkaloid 2 inhibited malate synthase from the pathogenic fungus Paracoccidioides spp. This enzyme is considered an important molecular target because it is not found in humans. Molecular docking simulations were used to describe the interactions between the alkaloids and malate synthase.
Yeast cells of the human pathogenic fungus Paracoccidioides brasiliensis strain Pb01 were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved by ...electroporation, with intact or linearized plasmid DNA. The fungus was transformed using 200 mM manitol, 5 or 7 kV/cm field strength, 25 µF capacitance, 400 resistance, 5 µg plasmid DNA and 107 yeast cells in 400 µl, and selected in BHI medium overlaid with 30 µg/ml hygromycin B (hygB). Mitotic stability was assessed by growing transformants on non-selective BHI medium, followed by plating on hygromycin B (30 µg/ml). Transformants were analyzed by PCR and Southern blotting, confirming the hph gene integration into the transformants genome. A low level of stability of the integrated hph sequence in the transformant genomes was observed, probably because of the multinuclearity of P. brasiliensis yeast cells.
The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of ...resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MS(E). The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied.
Approximately one-third of all proteins have been estimated to contain at least one metal cofactor, and these proteins are referred to as metalloproteins. These represent one of the most diverse ...classes of proteins, containing metal ions that bind to specific sites to perform catalytic, regulatory and structural functions. Bioinformatic tools have been developed to predict metalloproteins encoded by an organism based only on its genome sequence. Its function and the type of metal binder can also be predicted via a bioinformatics approach. Paracoccidioides complex includes termodimorphic pathogenic fungi that are found as saprobic mycelia in the environment and as yeast, the parasitic form, in host tissues. They are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis in Latin America. Many metalloproteins are important for the virulence of several pathogenic microorganisms. Accordingly, the present work aimed to predict the copper, iron and zinc proteins encoded by the genomes of three phylogenetic species of Paracoccidioides (Pb01, Pb03, and Pb18). The metalloproteins were identified using bioinformatics approaches based on structure, annotation and domains. Cu-, Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by Paracoccidioides spp. genomes. Zinc proteins were the most abundant metalloproteins, representing 5.7% of the fungus proteome, whereas copper and iron proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that metalloproteins are related to many cellular processes. Furthermore, it was observed that many of these metalloproteins serve as virulence factors in the biology of the fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the Paracoccidioides spp. are of the utmost importance for the biology and virulence of these particular human pathogens.
The genus
comprises known fungal pathogens of humans and can be isolated from different infection sites. Metabolic peculiarities in different members of the
led us to perform proteomic studies in the ...presence of the two-carbon molecule acetate, which predominates in the nutrient-poor environment of the phagosome. To investigate the expression rates of proteins of different members of
, including one isolate of
(
01) and three isolates of
(
03,
339, and
EPM83), using sodium acetate as a carbon source, proteins were quantified using label-free and data-independent liquid chromatography-mass spectrometry. Protein profiles of the isolates were statistically analyzed, revealing proteins that were differentially expressed when the fungus was cultivated in a non-preferential carbon source rather than glucose. A total of 1,160, 1,211, 1,280, and 1,462 proteins were reproducibly identified and relatively quantified in
and the
isolates
03,
339, and
EPM83, respectively. Notably, 526, 435, 744, and 747 proteins were differentially expressed among
and the
isolates
03,
339, and
EPM
, respectively, with a fold-change equal to or higher than 1.5. This analysis revealed that reorganization of metabolism occurred through the induction of proteins related to gluconeogenesis, glyoxylic/glyoxylate cycle, response to stress, and degradation of amino acids in the four isolates. The following differences were observed among the isolates: higher increases in the expression levels of proteins belonging to the TCA and respiratory chain in
EPM83 and
01; increase in ethanol production in
01; utilization of cell wall components for gluconeogenesis in
03 and
EPM83; and increased β-oxidation and methylcitrate cycle proteins in
01and
EPM83. Proteomic profiles indicated that the four isolates reorganized their metabolism in different manners to use acetate as a carbon source.
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